基于寨卡病毒NS2B蛋白特异抗原多肽的荧光素酶免疫吸附法检测寨卡病毒抗体
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摘要
目前国内外针对寨卡病毒(Zika virus,ZIKV)的研究多集中于E蛋白与NS1蛋白,而对于其他非结构蛋白的研究则相对较少。为了进一步了解ZIKV感染免疫机制,为ZIKV免疫学检测和流行病学研究提供新的工具方法,本研究建立了基于NS2B蛋白多肽抗原荧光素酶免疫吸附试验(Luciferase immunosorbent assay,LISA)的新方法,应用于检测抗ZIKV IgG抗体(anti-ZIKV IgG)。通过构建NS2B蛋白特异性多肽抗原与新型荧光素酶融合表达的载体,转染真核细胞后获得含有目标抗原与荧光素酶融合蛋白的细胞裂解物,建立anti-ZIKV IgG检测的LISA法。采用正常人血清、ZIKV感染的阳性血清、乙脑病毒(Japanese encephalitis virus,JEV)感染阳性血清,登革病毒(Dengue virus,DENV)感染阳性血清,评价该方法的特异性、灵敏度与重复性及交叉反应,并与基于NS1纯化抗原的商业化ELISA试剂盒(NS1-ELISA)进行平行比较。成功建立的NS2B-LISA方法,检测灵敏度和特异度分别为92%和96.2%,通过阳性样本倍比稀释评价两种检测方法灵敏度,NS2B-LISA的灵敏度比NS1-ELISA高16倍。重复性实验显示NS2B-LISA法检测anti-ZIKV IgG板间变异系数为(3.1±0.7)%,板内变异系数为(2.6±0.9)%。本研究基于NS2B蛋白多肽抗原成功建立了anti-ZIKV IgG检测的LISA新方法,该方法简便、特异、灵敏、重复性好,易于高通量自动化,可用于anti-ZIKV IgG感染的免疫学诊断与流行病学研究。
At present,the research on Zika virus(ZIKV)at home and abroad mostly focuses on E protein and NS1 protein,while the research on other non-structural proteins is relatively less. To further understand the immune response to ZIKV infection and provide new tools and methods for immunological detection and epidemiological study of ZIKV,we established a luciferase immunosorbent assay(LISA) based on NS2B antigen to detect IgG antibody against ZIKV. We constructed an expression vector carrying a fusion protein of NS2B-specific polypeptide and nano-luciferase. Cell lysate containing the fusion protein was obtained after transfecting eukaryotic cells,and the LISA method for anti-ZIKV IgG detection was assessed. The specificity,sensitivity and reproducibility of the method were evaluated using positive sera infected with ZIKV and normal human sera compared with a commercial ELISA kit based on NS1 antigen(NS1-ELISA). The NS2B-LISA method was successful,the sensitivity and specificity were 92% and 96.2%,respectively. The sensitivity of the two methods was evaluated based on serial dilution of positive sera. The sensitivity of the NS2B-LISA was 17 times that of NS1-ELISA. In tests of reproducibility of NS2B-LISA,the coefficient of variation of an anti-ZIKV IgG plate was(3.1±0.7)% and the intraplate variation coefficient was(2.6±0.9)%. Therefore,a novel LISA method for anti-ZIKV IgG detection was successfully established based on an NS2B peptide antigen,the method is simple,specific,sensitive,reproducible,and easy to automate with high throughput.This NS2B-LISA can be used for immunological diagnosis and epidemiological investigation of ZIKV infection.
At present,the research on Zika virus(ZIKV)at home and abroad mostly focuses on E protein and NS1 protein,while the research on other non-structural proteins is relatively less. To further understand the immune response to ZIKV infection and provide new tools and methods for immunological detection and epidemiological study of ZIKV,we established a luciferase immunosorbent assay(LISA) based on NS2B antigen to detect IgG antibody against ZIKV. We constructed an expression vector carrying a fusion protein of NS2B-specific polypeptide and nano-luciferase. Cell lysate containing the fusion protein was obtained after transfecting eukaryotic cells,and the LISA method for anti-ZIKV IgG detection was assessed. The specificity,sensitivity and reproducibility of the method were evaluated using positive sera infected with ZIKV and normal human sera compared with a commercial ELISA kit based on NS1 antigen(NS1-ELISA). The NS2B-LISA method was successful,the sensitivity and specificity were 92% and 96.2%,respectively. The sensitivity of the two methods was evaluated based on serial dilution of positive sera. The sensitivity of the NS2B-LISA was 17 times that of NS1-ELISA. In tests of reproducibility of NS2B-LISA,the coefficient of variation of an anti-ZIKV IgG plate was(3.1±0.7)% and the intraplate variation coefficient was(2.6±0.9)%. Therefore,a novel LISA method for anti-ZIKV IgG detection was successfully established based on an NS2B peptide antigen,the method is simple,specific,sensitive,reproducible,and easy to automate with high throughput.This NS2B-LISA can be used for immunological diagnosis and epidemiological investigation of ZIKV infection.
引文
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[12]Lustig Y,Zelena H,Venturi G,Van Esbroeck M,Rothe C,Perret C,Koren R,Katz-Likvornik S,Mendelson E,Schwartz E.Sensitivity and kinetics of an NS1-based Zika virus enzyme-linked immunosorbent assay in Zika virus-infected travelers from Israel,the Czech Republic,Italy,Belgium,Germany,and Chile[J].J Clin Microbiol,2017,55(6):1894-1901.
[13]Badshah S L,Naeem A,Mabkhot Y.The new high resolution crystal structure of NS2B-NS3 protease of Zika virus[J/OL].Viruses,2017,9(1).pii:E7
[14]Zhang Z,Li Y,Loh Y R,Phoo W W,Hung A W,Kang C,Luo D.Crystal structure of unlinked NS2B-NS3 protease from Zika virus[J].Science,2016,354(6319):1597-1600.
[2]Wiratsudakul A,Suparit P,Modchang C.Dynamics of Zika virus outbreaks:an overview of mathematical modeling approaches[J/OL].PeerJ,2018,6:e4526.
[3]Guedes D R,Paiva M H,Donato M M,Barbosa P P,Krokovsky L,Rocha S,Saraiva K,Crespo M M,Rezende T M,Wallau G L,Barbosa R M,Oliveira CM,Melo-Santos M A,Pena L,Cordeiro M T,Franca R,Oliveira A L,Peixoto C A,Leal W S,Ayres C F.Zika virus replication in the mosquito Culex quinquefasciatus in Brazil[J/OL].Emerg Microbes Infect,2017,6(8):e69.
[4]Styczynski A R,Malta J,Krow-Lucal E R,Percio J,Nobrega M E,Vargas A,Lanzieri T M,Leite P L,Staples J E,Fischer M X,Powers A M,Chang G J,Burns P L,Borland E M,Ledermann J P,Mossel E C,Schonberger L B,Belay E B,Salinas J L,Badaro R D,Sejvar J J,Coelho G E.Increased rates of Guillain-Barre syndrome associated with Zika virus outbreak in the Salvador metropolitan area,Brazil[J/OL].PLoS Negl Trop Dis,2017,11(8):e5869.
[5]Lanciotti R S,Kosoy O L,Laven J J,Velez J O,Lambert A J,Johnson A J,Stanfield S M,Duffy M R.Genetic and serologic properties of Zika virus associated with an epidemic,Yap State,Micronesia,2007[J].Emerg Infect Dis,2008,14(8):1232-1239.
[6]Mishra N,Caciula A,Price A,Thakkar R,Ng J,Chauhan L V,Jain K,Che X,Espinosa D A,Montoya C M,Balmaseda A,Sullivan E H,Patel J J,Jarman RG,Rakeman J L,Egan C T,Reusken C,Koopmans M,Harris E,Tokarz R,Briese T,Lipkin W I.Diagnosis of zika virus infection by peptide array and enzyme-linked immunosorbent assay[J/OL].MBio,2018,9(2).pii:e00095-18.
[7]Wang H,Cai Q,Liang Y,Shui J,Tang S.A simple and high-throughput luciferase immunosorbent assay for both qualitative and semi-quantitative detection of antiHIV-1 antibodies[J].Virus Res,2019,263:9-15.
[8]芜为,邢骁跃,张全福.寨卡病毒IgG抗体间接ELISA检测方法的初步建立[J].病毒学报,2017,33(2):253-257.DOI:10.13242/j.cnki.bingduxuebao.003130
[9]Zhang B,Pinsky B A,Ananta J S,Zhao S,Arulkumar S,Wan H,Sahoo M K,Abeynayake J,Waggoner J J,Hopes C,Tang M,Dai H.Diagnosis of Zika virus infection on a nanotechnology platform[J].Nat Med,2017,23(5):548-550.
[10]Talarmin A,Labeau B,Lelarge J,Sarthou J L.Immunoglobulin A-specific capture enzyme-linked immunosorbent assay for diagnosis of dengue fever[J].JClin Microbiol,1998,36(5):1189-1192.
[11]Steinhagen K,Probst C,Radzimski C,SchmidtChanasit J,Emmerich P,van Esbroeck M,Schinkel J,Grobusch M P,Goorhuis A,Warnecke J M,Lattwein E,Komorowski L,Deerberg A,Saschenbrecker S,Stocker W,Schlumberger W.Serodiagnosis of Zika virus(ZIKV)infections by a novel NS1-based ELISAdevoid of cross-reactivity with dengue virus antibodies:a multicohort study of assay performance,2015 to 2016[J].Euro Surveill,2016,21(50).pii:30426.
[12]Lustig Y,Zelena H,Venturi G,Van Esbroeck M,Rothe C,Perret C,Koren R,Katz-Likvornik S,Mendelson E,Schwartz E.Sensitivity and kinetics of an NS1-based Zika virus enzyme-linked immunosorbent assay in Zika virus-infected travelers from Israel,the Czech Republic,Italy,Belgium,Germany,and Chile[J].J Clin Microbiol,2017,55(6):1894-1901.
[13]Badshah S L,Naeem A,Mabkhot Y.The new high resolution crystal structure of NS2B-NS3 protease of Zika virus[J/OL].Viruses,2017,9(1).pii:E7
[14]Zhang Z,Li Y,Loh Y R,Phoo W W,Hung A W,Kang C,Luo D.Crystal structure of unlinked NS2B-NS3 protease from Zika virus[J].Science,2016,354(6319):1597-1600.